Standard: human intestine-on-a-chip

Organs-on-chips are microphysiological systems that allow to replicate the key functions of human organs and accelerate the innovation in life sciences including disease modeling, drug development, and precision medicine. However, due to the lack of standards in their definition, structural design, cell source, model construction, and functional validation, a wide range of translational application of organs-on-chips remains a challenging. “Organs-on-chips: Intestine” is the first group standard on human intestine-on-a-chip in China, jointly agreed and released by the experts from the Chinese Society of Biotechnology on 29th April 2024. This standard specifies the scope, terminology, definitions, technical requirements, detection methods, and quality control in building the human intestinal model on a chip. The publication of this group standard will guide the institutional establishment, acceptance and execution of proper practical protocols and accelerate the international standardization of intestine-on-a-chip for translational applications.


Scope
This document specifies the ethical requirements, Technical requirements, and detection methods for construction and evaluation of human intestine-on-a-chip.
This document is applicable to the construction, Detection and evaluation activities of functional intestine-on-a-chip

Normative references
The contents of the following documents are essential provisions of this document through normative reference.For referenced documents with specified dates, only the version corresponding to that date is applicable to this document; for documents without specified dates, the latest version (including all amendments) is applicable to this document:

Terms and definitions
The terms defined in T/CSB 0003-2024 and the following terms and definitions apply to this document:

Intestine-on-a-chip
An intestinal microphysiological system that can reflect the key structures and biofunctions (such as absorption, secretion, metabolism, and barrier properties) of human intestine by seeding multiple cells (e.g., intestinal epithelial cells and vascular endothelial cells) and reconstructing microenvironmental elements (e.g., tissue-tissue interface and fluid flow), or applying cyclic tension to mimic intestinal peristalsis (T/CSB 0003-2024, 5.5).

Intestinal barrier
The integration of structures and functions that ensure the efficient absorption of nutrients and barricade harmful substances (e.g., bacteria and toxins) from permeating through the intestinal mucosa into the body's organs and bloodstream (Chinese Terms in Parenteral and Enteral Nutrition-2019).

Intestinal villus
Tiny projections on the surface of the mucous membrane of the small intestine, of which a single layer of columnar epithelium is on the surface and the mucosal lamina propria is in the axis (Chinese Terms in Histology and Embryology (2nd Edition)-2014).

Intestinal microenvironment
The summation of biophysical and biochemical factors in intestinal tissue that affect cell growth and functions, including various cell types, extracellular matrix, fluid stimuli, physicochemical gradients and intestinal flora (Kim 2012;Guo 2018).

Intestinal epithelial cells
Single-layered cells that make up the luminal surface (inner layer) of the gastrointestinal tract, including the small intestine and colon.Intestinal epithelial cells of the small intestine include Paneth cells and undifferentiated cells (Shin 2018).

Organoid
Self-assembled 3D microtissues derived from stem cells or organ-specific precursor cells through self-renewal, differentiation, and self-organization, which consist of multiple cell types and can partially reflect the key structures and specific functions of their source tissue or organ (T/CSB 0003-2024, 3.3).

Intestinal organoid
Self-assembled intestinal microtissues derived from stem cells through self-renewal, differentiation, and selforganization, which consist of multiple types of mature intestinal epithelial cells and can partially reflect the key structures and tissue-specific functions of in vivo intestinal epithelium.

Intestinal barrier permeability
The ability of compounds to pass through the intestinal barrier.Intestinal barrier permeability indirectly impacts the absorption of compounds in the body, focusing on the fraction of the absorbed dose rather than systemic bioavailability.It is directly linked to the rate at which compounds transfer across the intestinal barrier (Kwon 2021).

Bioavailability
The relative amount and rate at which a bioactive substance (such as drugs, nutrients, or toxins) is absorbed into the body's circulation, depending on various factors, including intestinal barrier permeability, the solubility of the substance, its molecular size, and the presence of transporters.Bioavailability reflects the information related to the time and dose at which substances enter the bloodstream, tissue organ uptake, and pharmacological effects (Yang and Chen 2018).

Transepithelial electrical resistance, TEER
The resistance to ion flow across cell layers.TEER is related to the integrity of tight junctions between cells and can reflect the integrity and permeability of the cellular barrier (Odijk 2015;van der Helm 2016;Guo 2024).

Apparent permeability coefficient, Papp
The relative rate at which compounds pass through the intestinal barrier per unit area of the intestine over a certain period.Papp is an important indicator for assessing the absorption of compounds in the intestine.
The formula for calculating the apparent permeability coefficient is Papp = (dQ/dt) / (A × c 0 ), where: -(dQ/dt) is the amount of compound passing through the intestinal barrier per unit time, -A is the surface area of the intestine, -c 0 is the initial concentration of the compound.

General principles
When the preparation and evaluation process of an intestine-on-a-chip involves the use of human primary tissues or cells, which raises ethical concerns, ethical approval should be obtained, and informed consent should be obtained to ensure the privacy protection of the donors.

Informed consent
Prior to the collection of human samples, written informed consent should be obtained from the donors, clearly outlining the rights and responsibilities of both the donors and the sample collection entity.Informed consent should comply with the requirements of Sect.5.3 of GB/T 38736-2020.

Ethical review
The establishment and research protocols of the intestinal chip should be reviewed and approved by the ethics review committee of the project's sponsoring and primary executing institution.

Privacy protection
Human sample donors have the right to personal privacy, and the confidentiality and protection of their personal privacy information should comply with the requirements of Chapter 6 of GB/T 38736-2020.

Fabrication of the chip
Design of the chip The structure of the intestinal chip carrier typically includes two chambers separated by a porous membrane: one chamber for seeding intestinal epithelial cells and the other chamber for seeding vascular endothelial cells, with fluid environments present in both chambers (Li 2017).
Note 1: The structure of the intestinal chip carrier can also take other reasonable forms, such as using extracellular matrix instead of a porous membrane.
Note 2: Depending on the experimental purposes, other cells such as immune cells may also be added to the chambers on both sides of the porous membrane.
Material selection for the chip The commonly used materials for the intestine-on-a-chip should preferably be PDMS.
Other materials for the intestine-on-a-chip typically include PMMA, PS, and PC.
The material of the porous membrane can be selected from PDMS, PC, PET, and nylon, with pore sizes ranging from 0.01 μm to 10 μm.
Methods for chip fabrication PDMS chips are commonly fabricated using soft lithography method.
Chips made of materials such as PMMA, PS, and PC are preferably manufactured using techniques such as injection molding, machining and 3D printing.
For PDMS chips, plasma bonding is typically used for processing; for plastic materials such as PMMA, PS, and PC, methods such as heat sealing, ultrasonic sealing, and adhesive sealing are commonly employed.

Quality control for chip
The cytotoxicity of chips should be tested using methods outlined in GB/T 16886.5-2017.The chips should meet the non-cytotoxicity requirements outlined in Chapter 8 of GB/T 16886.5-2017.

Seeding cells in the chip
Cell components Intestine-on-a-chip should contain human intestinal epithelial cells (Guo 2021).
Note: Intestinal epithelial cells typically include Caco-2 cell line, primary human intestinal cells, or intestinal organoids, among others.
When simulating the structure and function of the intestinal epithelial-endothelial barrier, intestinal chips should include endothelial cells.
Note: Endothelial cells should preferably be derived from human umbilical vein endothelial cells, human intestinal microvascular endothelial cells, or endothelial cells differentiated from stem cells (such as induced pluripotent stem cells and embryonic stem cells), with a maximum passage number of 10.
When simulating intestinal immune function, immune cells should be added to the endothelial cell side of the intestinal chip.
Note: Immune cells typically include monocytes and macrophages, among others.Testing should follow the "Sterility Test" method outlined in the "Pharmacopoeia of the People's Republic of China (2020 Edition)."After culturing by membrane filtration or direct inoculation for no less than 14 days, observe whether the culture medium becomes turbid, or take culture fluid smears, stain, and microscopically examine.

Requirements of cell performance
ii) Chlamydia Testing should follow the "Chlamydia Test" method outlined in the "Pharmacopoeia of the People's Republic of China (2020 Edition)."Testing can be done using Chlamydia culture method or indicator cell culture method (DNA staining), or other methods approved by the national drug regulatory authority.
iii) HIV Testing should be conducted using the WS 293 nucleic acid method, with HIV-related RNA below the detection limit in routine PCR.iv) HBV Testing should be conducted using the WS 299 nucleic acid method, with HBV-related RNA below the detection limit in routine PCR.
v) HCV Testing should be conducted using the WS 213 nucleic acid method, with HCV-related RNA below the detection limit in routine PCR.
vi) Exogenous Viral Factors Testing should follow the "Exogenous Viral Factor Test" method outlined in the "Pharmacopoeia of the People's Republic of China (2020 Edition)," using methods such as cell culture, inoculation of suckling mice or chicken embryos, nucleic acid amplification technology, etc.
When human stem cells are used in the chip, they should meet the requirements of Chapter 5 and Chapter 6 of T/CSCB 0001-2020, as well as Chapter 8 of GB/T 42466-2023.
When intestinal organoids are used in the chip, the cellular composition and proportions of the organoids should comply with the requirements of Sect.6.4 of T/ CSCB 0013-2022 (Wang 2023).

Dynamic culture in the chip
Perfusion in the chip Fluid flow in the intestinal chip for cell culture can be achieved using syringe pumps, peristaltic pumps, or gravity-driven methods to meet the requirements for dynamic cell culture conditions.

Range of the shear stress
The range of shear stress generated by the fluid in the chip culture medium typically ranges from 0.001 dyn/cm 2 to 10 dyn/cm 2 .

Method for chip fabrication
The soft lithography method for preparing the intestinal chip carrier is detailed in Appendix A.

Method for seeding cells in the chip
The method for seeding cells in the intestine-on-a-chip is detailed in Appendix B, sections B.3.1 to B.3.3.

Method for culturing cells in the chip
Cell culture on the intestinal chip should comply with the provisions detailed in Appendix B, section B.3.4.The calculation method for fluid shear stress in the intestinal chip should comply with the provisions detailed in Appendix C.

Intestinal villi morphology
After applying fluid shear stress to the intestinal organ chip for a certain period, dark arc-shaped, tubular, or circular villous structures should be observed, with clear boundaries.

Tissue barrier function
Immunofluorescence characterization In the intestineon-a-chip, the expression of one or more tight junction proteins such as ZO-1, Occludin, and Claudin-1 in epithelial cells should generally be detected.
TEER measurement TEER values should typically be measured when the intestinal organ chip cells have cultured to form a complete intestinal barrier: a) When only the Caco-2 cell line is present in the intestinal organ chip, the TEER value is usually greater than 500 Ω•cm 2 .b) When co-cultured Caco-2 and HT-29-MTX cells are present in the intestinal organ chip, the TEER value is typically greater than 100 Ω•cm 2 .c) When only organoid-derived cells are present in the intestinal organ chip, the TEER value is typically greater than 200 Ω•cm 2 .
Intestinal barrier permeability After the tissue barrier is fully formed in the intestinal organ chip, permeability is determined using 4 kDa dextran (Chen 2018).Generally, the apparent permeability coefficient (Papp) value should be less than 1 × 10 -6 (1 h).

Specific genes expression in chip
Intestinal organ chips typically should detect the expression of marker genes ALPI (Intestinal Alkaline Phosphatase) and Villin, which are indicative of absorptive intestinal epithelial cells.When goblet cells are present in the intestinal chip, the marker gene MUC2 (Mucin 2) should also be detected (Kwon 2021).

Functional protein secretion in chip
When goblet cells are present in the intestinal organ chip, secretion of mucin MUC2 is typically detected.

Observation of intestinal villi
Bright-field observation During the cell culture process of the intestinal organ chip, observation should be conducted daily under an inverted phase contrast microscope to determine the appearance and morphological changes of intestinal villi.
SEM observation SEM observation of intestinal villi in the intestinal organ chip should comply with the methods outlined in Appendix D.

Assessment of intestinal barrier
Immunofluorescence characterization After the formation of the intestinal tissue barrier in the intestinal organ chip, tight junctions between cells should be visible under a microscope, with clear cell boundaries.Detection of the expression of tight junction proteins in the tissue barrier of the intestinal organ chip should comply with the methods outlined in Appendix E.
TEER measurement TEER measurement is mainly based on Ohm's law and electrochemical impedance methods: When measuring TEER based on Ohm's law, alternating current square waves are typically applied to the cells to maintain a constant small current.
When measuring TEER using the electrochemical impedance method, working electrodes and counter electrodes are typically placed on both sides of the cell layer, and the impedance of the measurement system is measured.
Molecular permeability detection Molecular permeability testing of the intestinal organ chip tissue barrier should comply with the methods outlined in Appendix F, section F.3.The data processing process should comply with the methods outlined in Appendix F, sections F.4 to F.6.

D.3.1 Cell Fixation
After a specified period of cell culture in the intestinal organ chip, remove the culture medium and wash the cells with buffer solution.Then, add an appropriate volume of tissue fixative (D.2.4) to fully immerse the cells in the intestinal organ chip.Once the cells are completely fixed, thoroughly rinse them with buffer solution in preparation for the next experiment.D.3.2 Dehydration Sequentially dehydrate the cells in the chip by immersing them in ethanol (D.2.2) solutions with the following volume concentrations: 30%, 50%, 70%, 80%, 90%, 95%, and two rounds of 100%, all at room temperature.D.3.3 Sample Drying Transfer the samples from step A.3.2 into the critical point dryer (D.1.1)and dry them according to the standard procedure for a minimum of 2 h.D.3.4 Sample Coating Once the cell samples are thoroughly dried, utilize the vacuum ion sputtering instrument (D.1.2) to apply a thin layer of carbon, gold, or platinum onto the surface of the samples.This coating is essential to enhance their conductivity.D.3.5 SEM Observation SEM (D.1.3)can be employed to examine the microvilli structure on the surface of the samples after collecting image data.once with HBSS buffer flowing through the chip.Subsequently, replace the medium flowing through the chip with the prepared 1 mg/ml FITC-Dextran solution for perfusion incubation.F.3.2 Drawing the Standard Curve Prepare a standard curve of concentration versus fluorescence intensity by diluting the FITC-Dextran solution in HBSS buffer in a 96-well plate with a certain concentration gradient and determine the linear range.F.3.3 Fluorescence Quantification After 1 h of cell incubation, take a certain amount of effluent from the porous membrane into a 96-well plate and measure the fluorescence intensity of Dextran using a microplate reader.

F.4 Calculation of Apparent Permeability Coefficient
Calculating the apparent permeability coefficient using Formula (2): where: -Papp is the apparent permeability coefficient, with units of cm/s.-dQ is the total amount of Dextran permeated in a certain time, with units of mg.-dt is the time of the experiment, with units of s.
-A is the membrane area of the chip, with units of cm 2 .-c 0 is the initial concentration of Dextran, with units of mg/ml.

F.5 Calculation and Analysis
Repeat the experiment, calculate the average of three times, and record it as the apparent permeability coefficient of Dextran in the chip at that molecular weight.
F.6 Result Analysis For a processing time of 1 h, the Papp value for 4 kDa Dextran should be less than 1 × 10 -6 for a complete intestinal barrier.The larger the molecular weight, the smaller the Papp value.
GB/T 16886.5-2017Biological evaluation of medical devices-Part 5: Tests for in vitro cytotoxicity GB/T 38736-2020 Ethical Requirements of Human Biobanking GB/T 42466-2023 Technical Specification for Pluripotent Stem Cells Management of Biobanking WS 213 Diagnostic for Hepatitis C WS 293 Diagnostic Criteria for HIV/AIDS WS 299 Diagnostic Criteria for Viral Hepatitis B T/CSB 0003-2024 General Terminology of Organson-chips T/CSCB 0001-2020 General Requirements for Stem Cells T/CSCB 0013-2022 Human Intestinal Organoid The typical requirements for cell performance in the chip include: a) Cell Morphology The morphology of various cell types before seeding should correspond to their normal morphology, which can be referenced on the ATCC website.For example:-Caco-2 cells exhibit cuboidal, epithelial-like, or giantcell-like morphology, with some cells containing large vacuoles.-HT-29-MTX cells are round or square-shaped and tend to grow in clusters.-Intestinal organoids appear as spheroids or budlike structures with a central lumen, surrounded by tightly packed columnar epithelial cells.-Endothelial cells have flat polygonal or spindleshaped morphology.-In immune cells, monocytes often have kidney-shaped or horseshoe-shaped nuclei, with varying cell shapes including round and polygonal; macrophages are round or elliptical, with short projections, and activated ones may extend irregularly-shaped pseudopods.b) Cell Chromosome Karyotype The normal chromosome karyotype of cells should be 46, XY or 46, XX. c) Microbiological Testing Microbiological testing indicators include: i) Bacteria and Fungi Detection of cell marker gene expression in the intestinal organ chip should comply with the methods outlined in Appendix G, sections G.3.1 to G.3.3.The data processing process should comply with the methods outlined in Appendix G, section G.3.4.D.2.3 Phosphate Buffered Saline (PBS): Main components include Na2HPO4, KH2PO4, NaCl, and KCl, with a pH of 7.2 ~ 7.4.D.2.4 Tissue Fixative: 4% paraformaldehyde solution.D.2.5 0.1 M Phosphate Buffer (PB): Main components are KH2PO4 and NaOH, with a pH of 7.4.